Glaucoma is the number one cause of acquired blindness in adults in Japan, affecting an average of approximately 5% of those at least 40 years old. The definition of glaucoma is “a disease characterized by functional, structural abnormality of the eye having characteristic changes in the optic nerve and visual field in which optic nerve damage can usually be improved or suppressed by lowering the intraocular pressure sufficiently” (Non-patent Reference 1). Glaucoma is broadly classified as I) primary glaucoma, II) secondary glaucoma, and III) developmental glaucoma. I) primary glaucoma is also classified as 1) glaucoma primary open-angle glaucoma (broad sense) (A: primary open-angle glaucoma, B: normotensive glaucoma), 2) primary closed-angle glaucoma (A: primary closed-angle glaucoma, B: iris plateau glaucoma), and 3) mixed glaucoma (Non-patent Reference 1).
Elevated intraocular pressure is one finding often seen in glaucoma, but in Japan normotensive glaucoma in which the intraocular pressure remains constantly at a normal value during the course of development and progress of the glaucoma accounts for approximately 70% of all glaucoma patients. This is a trend seen conspicuously in Asia. The treatment method for glaucoma currently relied upon, however, is only lowering the intraocular pressure, and treatment methods for factors other than intraocular pressure are being sought.
Glaucoma in all its forms is characterized by the progressive disappearance of retinal ganglion cells (RGC) and corresponding visual field abnormalities. The disappearance of RGC has recently been suggested to be affected by calpain activity, and disappearance of RGC is reported to be decreased by administration of a calpain inhibitor in an animal model of normotensive glaucoma (Non-patent Reference 2).
The participation of calpains is also suggested in retinitis pigmentosa (RP), age-related macular degeneration (AMD), and retinal neuropathy associated with diabetic retinopathy (Non-patent References 3-6).
Further, it is thought that calpain activation participates in retinal vascular occlusive diseases such as retinal vein occlusion and retinal artery occlusion, which are ischemic diseases (Non-patent References 7 and 8).
Calpains are cysteine proteases that act Ca2+-dependently. They are expressed universally in the central nervous system and are related to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Calpains are expressed by RGC and the nerve fiber layer in the retina (Non-patent References 9-11), and activation of calpains is confirmed in the GCL (ganglion cell layer) of retinal grafts after axotomy and in rat glaucoma models (Non-patent References 12 and 13). Calpains indirectly induce an apoptosis pathway, and, as a result, are related to the disappearance of RGC.
From this viewpoint, it is thought that being able to observe calpain activity in real time in animal models would make it possible to:
(1) clarify when, where, and how calpain activity participates in the pathology of retinal diseases and
(2) use the knowledge of (1) to assess the progress of retinal diseases and the timing of treatment intervention.
Calpain probes reported to date are:
(A) a protein-based probe in which a fluorescent protein domain is bonded to a peptide that serves as a substrate of calpain (Non-patent Reference 14) and
(B) a probe in which a dipeptide is bonded to coumarin (Non-patent Reference 15). However, the development of a calpain activity-detecting fluorescent probe that does not rely on gene introduction and does not use UV light for excitation light is required in imaging in live cells.